Mass spectrometers are often coupled with chromatography systems in order to identify and characterize components of interest in a test sample. In such a coupled system, the eluting components from a chromatographic system are ionized in a mass spectrometer and a series of mass spectra are obtained at small time intervals, ranging from, for example, 0.01-10 seconds, for the duration of the chromatographic process. Each mass spectrum records the m/z values and intensities for all ions detected at each time point along the chromatographic time scale. As a test sample may contain many components (i.e., chemical entities), it is challenging to identify the components of interest amid complex mixtures in the resultant data.
The issue of signals arising from sample matrix components is the major confounding factor to the identification of components of interest in a complex sample. Other factors that may confound the analysis include random instrument noise and chemical background. Typically, the random instrument noise in a modern high resolution instrument, e.g., a Fourier transform type of instrument, is at low intensity levels and is not a primary concern. In addition, there have been a number of algorithms developed over the years to deal with noise, including Component Detection Algorithm (CODA) [Windig W, Payne A W. U.S. Pat. No. 5,672,869, Sep. 30, 1997], Sequential Paired Covariance [Muddiman D C, Rockwood A L, Gao Q, Severs J C, Udseth H R, Smith R D. Anal. Chem. 1995; 67: 4371], and Windowed Mass Selection Method [Fleming C M, Kowalski B R, Apffel A, Hancock W S. J. Chromatogr. A, 1999; 849: 71-85], for example. Chemical background signals are typically originated from solvents, column residues, and ion source contaminants; and they are typically common to all samples in an analysis.
In order to illustrate the major issue of sample matrix components to LC/MS analysis of complex samples, base peak ion chromatograms of an example discussed hereafter are shown in FIGS. 1a and 1b. A base peak ion (BPI) chromatogram presents a profile consisting of signals of the most intense ion in each mass spectrum acquired along the chromatographic time scale. The BPI chromatogram in FIG. 1a is obtained from LC/MS analysis of a human liver microsomal incubation sample containing buspirone metabolites. This is a relatively simple in vitro sample without significant sample matrix-related signals to alter the profile of the buspirone metabolite peaks. Chemical background and random instrument noise are present at such insignificant levels that they do not affect the profile. Therefore, all distinct peaks exhibited in FIG. 1a represent buspirone and its metabolites. However, when the same buspirone metabolite sample was reconstituted into a complex sample matrix, e.g., human plasma, the resultant BPI chromatogram (FIG. 1b) is complicated by additional peaks arising from sample matrix components, making the identification of buspirone metabolite peaks difficult.
A variety of approaches and data acquisition & analysis software associated with mass spectrometers have been developed to identify components of interest in complex samples. Some approaches target a specific behavior or property of potential components of interest in either the data acquisition stage or the data analysis stage to facilitate their identification (e.g., based on their molecular ion masses or fragmentation patterns, as known in the art). However, such approaches may miss potential components of interest that deviate from the targeted behavior or property. An alternative approach is background subtraction, by which signals arising from sample matrix components as well as chemical background are checked and subtracted from the data of a test sample based on their presence in a control sample [Ueno T, Sueyoshi T, Tanaka E, Jinkawa R, Hamada A, Takegami Y. Shitsuryo Bunseki 1974; 22, 109-114] [Goodley P, Imitani K, Am. Lab, 1993; 25, 36B-36D]. In reality, however, the task of background subtraction is significantly complicated and difficult to implement in mass spectrometric applications where chromatography is involved.
Many vendors of mass spectrometer and software systems provide background subtraction or similar functions. As a typical example, Thermo Fisher Scientific markets a background subtraction tool which is based on a scan-for-scan spectral subtraction operation for data between the test and control samples at each chromatographic time point. (A scan here refers to a time event at which a mass spectrum is acquired.) It also offers options to specify a time window around the time point of each mass spectrum of the test sample to search for a suitable background spectrum in the corresponding control sample data or to average the control spectra within the time window into one background spectrum before performing the subtraction operation. As another typical example, Waters Corporation markets a Control Sample Comparison tool where extracted ion chromatograms are generated at a user-specified mass width stepping throughout the mass range for both the test and control sample data. Extracted ion chromatograms between the test and control samples are compared at each mass width step for the identification of prominent peaks in the test sample.
The above mentioned background subtraction functions can provide adequate results in applications of relatively simple samples including some in vitro sample analysis where components of interest are major and may be detected fairly easily. However, when dealing with more complex samples such as biological fluids, (e.g., urine or plasma extracts) or complex mixtures (e.g., impurity analysis for drug products formulated in polymeric emulsifiers), a multitude of sample matrix components may be encountered whose signals are often dominant and whose masses fall in a range such that isobaric interferences (i.e., of the same nominal m/z values) to the components of interest are almost always observed. In addition, the temporal variability of sample matrix components (i.e., their chromatographic time fluctuations between runs) are often difficult to control because of the matrix effect caused by differing amounts of sample matrix components loaded on a chromatography system.
For the scan-for-scan based background subtraction tools, the main problem is the chromatographic time fluctuations of components between the control and test samples, which prevents thorough removal of signals of chemical background and sample matrix components. The issue of chromatographic time fluctuations remains even with the option of specifying a time window to search for a suitable background spectrum. This is because components may behave differently from each other in terms of their temporal variability and there may not be a suitable spectrum to represent the diversity of chromatographic time fluctuations for all components in question. In addition, the option of spectral averaging seems to cause data degeneration and further impairs the background subtraction for complex samples. For the Control Sample Comparison tools, the comparison is done in an indirect way by first converting the data to extracted ion chromatograms and then comparing peaks formed in the chromatograms. This indirect approach is quite complicated and involves peak definition, smoothing, integration, defining a threshold value and some other parameters. In addition, the rendering of the data to extracted ion chromatograms at arbitrary mass widths may intrinsically cause some data degeneration. For example, isobaric interferences of sample matrix components may be overwhelming and overshadow peaks of components of interest. This may be partially alleviated by generating extracted ion chromatograms at a narrower mass width for data obtained from a high resolution mass spectrometer (e.g., a Time-of-Flight or Fourier Transform type of instrument). However, since the steps of mass widths are systematically set throughout the mass range, they may not be optimally set around the exact masses of components in the samples and still cause inaccurate chromatographic profiling and data comparison for complex samples. An additional disadvantage of such extracted ion chromatogram-based approach is that the processed results typically can only be viewed with special vendor-provided browsers and cannot be verified by ways of BPI chromatogram or total ion chromatogram and the associated spectral examination that are common practices for the examination of mass spectrometric data, as known in the art.
It will be appreciated that the diversity of chromatographic time fluctuations of components should be taken into consideration to allow for thorough removal of signals arising from extraneous components in a sample. It will also be appreciated that the precise comparison of components in a test sample against those in the control samples with the un-degenerated exact mass data is of importance for correct identification and subtraction of extraneous components. Accordingly, the present invention provides improved methods for background subtraction using control samples. A precisely and thoroughly background-subtracted data would allow for the detection of components of interest in complex samples.
In sample analysis with a chromatography/mass spectrometry system, it is desirable to not only identify the molecular ions for components of interest but also to obtain their fragment ion spectra for structural characterization. Typically a fragment ion spectrum is obtained in a tandem mass spectrometry (MS/MS) mode where a specific precursor ion (typically the molecular ion of a component) is selected and activated by a collision-induced disassociation process (CID), followed by subsequent analysis of the product ions (i.e., fragment ions) formed. Vendors of a number of mass spectrometer systems provide real time data-dependent MS/MS acquisition functions to allow for automatic generation of product ion spectra (i.e., fragment ion spectra) for certain precursor ions. In a data-dependent MS/MS acquisition approach, precursor ions can be limited to certain components of interest relying on a use-and-inclusion list or by using more specific survey scans such as neutral loss, precursor and enhanced multiply-charged scans, as known in the art. However, these approaches presume some knowledge of the components of interest, which is not always the case. Alternatively, a dynamic background signal exclusion process [Le Blanc, U.S. Pat. No. 7,351,956 B2, Apr. 1, 2008] can be used to obtain MS/MS spectra for more components in a sample. Although this approach can generate MS/MS spectra for a multitude of components in a complex sample, it lacks the ability to differentiate whether they are of interest or not.
It is known in the art that fragment ions may also be obtained using in-source fragmentation techniques that activate all ions in the ion source instead of activating only specific precursor ions. For example, Thermo Scientific markets a source CID technique for some of its instruments by which ion fragmentation occurs between the skimmer and the first multipole region for all ions passing through the region. Alternatively, Clayton et al reported a low-and-high collision energy switching technique on a quadrupole time-of-flight mass spectrometer [Clayton, E.; Bateman, R. H.; Preece, S.; Sinclair, I. Advances in Mass Spectrometry (2001), 15, 403-404.] to obtain both a molecular ion data set at low collision energy and a fragment ion data set at high energy. Both of the above mentioned CID techniques are conducted in non-selective manner as oppose to the foregoing described CID processes conducted in MS/MS mode. The advantage of non-selective CID techniques is that they generate fragment ions for all precursor ions formed in the ion source without missing anyone. However, the problem of non-selective CID techniques is that fragment ions generated may not be easily assigned to a precursor ion due to the non-specific nature of the CID activation, thus making the fragment ion information useless for elucidating the structure of a precursor ion of interest.
It will be appreciated that fragment ions from extraneous precursor ions should be removed so that relevant fragment ion information can be correctly assigned to the components of interest for structural elucidation. A precisely and thoroughly background-subtracted data should allow for the removal of extraneous fragment ion signals arising from chemical background and sample matrix components so that clean fragment ion spectra (also known as product ion spectra) comprising mainly relevant fragment ion information can be obtained for components of interest.